Journal: bioRxiv

Article Title: PRIME: A modular plasmid-based platform for continuous in vivo evolution across bacteria

doi: 10.64898/2026.09.28.754915

Figure Lengend Snippet: (a) The PRD1 bacteriophage possesses a 14927 bp genome encoding four proteins involved in protein-primed DNA replication: the DNA polymerase P1, the terminal protein P8, and the single-stranded DNA-binding proteins P12 and P19. (b) PRIME comprises four plasmids: pORM ( O rthogonal R eplication M achinery), pOTH ( O rthogonal T ransformation H elper), pORFITR ( O rthogonal R eplicons F lanked by Inverted T erminal R epeats), and lopEG ( L inear O rthogonal P lasmid with E volving G enes). The pORM plasmid carries the genes required for protein-primed DNA replication under control of the inducible P Cin promoter. P12_T2 and P19 are indicated in brackets, as these proteins are dispensable for orthogonal plasmid replication but are present in the pORM_P12_T2 construct. pORM additionally contains the broad-host-range pBBR1 origin of replication, enabling propagation in a wide range of bacterial hosts. The pOTH plasmid encodes accessory proteins facilitating lopEG transformation. Owing to their toxicity, these proteins are expressed under control of the inducible P BAD promoter. The temperature-sensitive pSC101(ts) origin enables efficient curing of pOTH following lopEG establishment. The pORFITR plasmid facilitates cloning and production of lopEG plasmids. It contains two inverted terminal repeat (ITR) regions flanked by MlyI recognition sites. As a Type IIS restriction enzyme, MlyI cleaves outside of its recognition sequence, enabling blunt cleavage precisely at the edges of the ITRs. The R6K conditional origin of replication prevents plasmid propagation in cells lacking the Pir protein, allowing direct transformation of lopEG plasmids without gel purification. (c) Workflow for establishment of orthogonal plasmids in E. coli . (1) Cells are first transformed with pORM and pOTH. (2) Expression of the replication proteins is induced, and the temperature is increased prior to lopEG transformation to inhibit further replication of pOTH. (3) lopEG plasmids are electroporated into competent cells expressing the PRD1 replication machinery. Orthogonal plasmid copy number can subsequently be tuned using the OHC14-HSL inducer. (d) PRIME enables continuous directed evolution experiments. Replacement of the wild-type P1 polymerase (green) with an error-prone variant (red) promotes the generation of DNA libraries directly in vivo without the need for repeated manual intervention. Iterative cycles of diversification and selection subsequently enable the rapid evolution of constructs with improved properties.

Article Snippet: The YFP gene was then replaced with the operon encoding for the orthogonal replication machinery by digestion with BsaI-HF-v2 and KpnI-HF restriction enzymes (New England Biolabs) and ligation with a similarly digested insert, yielding pORM_P12_P19.

Techniques: DNA Binding Assay, Plasmid Preparation, Control, Construct, Transformation Assay, Toxicity, Cloning, Sequencing, Gel Purification, Expressing, Variant Assay, In Vivo, Selection, Protein Binding